Pseudomonas putida with both p-xylene and nitrogen removal under anaerobic conditions, bacterial agent and method and application thereof

Abstract

The application provides a Pseudomonas putida with p-xylene and nitrogen removal under anaerobic conditions, a bacterial agent and a method and application thereof, and relates to the field of microbial fermentation. The application provides a Pseudomonas putida with p-xylene and nitrogen removal under anaerobic conditions, which has a preservation number of CCTCC NO: M20241686 and is preserved in the China Center for Type Culture Collection on July 25, 2024. The strain can remove NH4 + -N, NO3 ‑ -N and NO2 ‑ -N as a single nitrogen source (15 mg / L), the removal rates of NH4 + -N, NO3 ‑ -N and NO2 ‑ -N are 59.08%, 96.90% and 100% respectively; and when p-xylene is used as the only carbon source, the removal rate of the p-xylene of the strain can reach 55.54% within 72 hours. The optimal denitrification conditions of the strain are as follows: the optimal carbon source is sodium succinate hexahydrate, the optimal C / N is 3, the optimal culture temperature is 30 DEG C, the optimal pH is 7, the strain can tolerate weak alkaline environment, and the strain is not resistant to acidic environment.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation, and relates to *Pseudomonas putida*, specifically to *Pseudomonas putida* capable of removing both xylene and nitrogen under anaerobic conditions, its inoculum, methods, and applications. Background Technology

[0002] NO3 in water - Excessive NO3 concentrations can lead to a range of human health and environmental risks. - -N wastewater entering rivers and lakes stimulates the rampant growth of algae, leading to water quality deterioration, reduced biodiversity, and ecosystem degradation. - The toxicity of NO3- to livestock is of great concern, particularly in feed or groundwater. - Excessive NO3- concentrations can lead to increased mortality rates in livestock. In ruminants, NO3-... - -N is reduced to NO2 - -N can cause methemoglobinemia, manifesting as cyanosis and hypoxia, which immediately threatens the lives of ruminants, and hypoxia can cause miscarriage in pregnant ruminants. Therefore, groundwater quality standards stipulate that NO3-... - -N content must not exceed 20 mg / L. NO3 - -N can be used as a nutrient by algae when NO3 in the water... - When the -N content exceeds the water body's tolerance range, it may cause large-scale algal blooms.

[0003] Aromatic VOCs are commonly found in coal tar, petroleum products, and various organic chemical formulations. They are a typical pollutant found in chemically contaminated sites, with refineries, pesticide plants, chemical plants, gas stations, and composting plants being the main sources. Data shows that most volatile organic compounds in the environment are trace organic compounds with very low concentrations; nearly half of the VOCs concentrations are between 0.01 and 1.0 μg / L, some even below the detection limit. Although the concentration of VOCs in the environment is not high, due to their irritant and toxic properties, they are priority pollutants for water quality monitoring in my country.

[0004] Aromatic VOCs are diverse, including benzene, toluene, naphthalene, and nitrobenzene. Among them, paraxylene, a typical VOC, is widely used in the production of plastics, rubber, paints, and synthetic fibers. It is toxic, volatile, insoluble in water, and harmful to skin, eyes, respiratory system, and various organs. Paraxylene is an important chemical raw material in the petrochemical and pharmaceutical industries, used in the production of many polymers, plastics, solvents, and fuels. The International Tanker Owners Pollution Consortium has identified paraxylene as one of the 20 most hazardous and toxic chemicals. Paraxylene is widely used as an industrial solvent, exhibiting high fluidity in the environment in gaseous, liquid, or solid states. It can also be distributed as a pollutant in the environments of various industries, such as leather, paints, rubber and printing, gasoline, and aviation fuel. According to the "Integrated Emission Standard of Air Pollutants in China" (GB 16297-1996), the maximum allowable emission concentration in the atmosphere is 90 mg / m³. 3 Because paraxylene is a liquid at room temperature, it can enter surface water and soil. The "Surface Water Environmental Quality Standard" (GB 3838-2002) sets the xylene limit for centralized drinking water surface water sources at 0.5 mg / L. Paraxylene evaporates easily, and high concentrations are rarely found in surface water or topsoil. However, any paraxylene that does not evaporate from the topsoil can seep into groundwater. Therefore, eliminating paraxylene is crucial for ecosystems and public health and safety.

[0005] Biological nitrogen removal, commonly used in wastewater treatment systems, is more environmentally friendly, economical, and efficient compared to physicochemical nitrogen removal methods. Conventional biological nitrogen removal involves two processes: aerobic nitrification and anaerobic denitrification, carried out by nitrifying and denitrifying bacteria, respectively. Denitrification is the key to biological nitrogen removal technology. Denitrification reactions can be classified into three types based on nutrient type and oxygen demand: heterotrophic anoxic denitrification, heterotrophic aerobic denitrification, and autotrophic denitrification.

[0006] Eutrophication of water bodies leads to the regeneration of inorganic nutrients at the bottom, consuming oxygen and increasing primary productivity. The growth and decay of primary producers in the water create organic matter, whose rapid degradation consumes dissolved oxygen (DO), resulting in an anaerobic environment. Facultative anaerobic bacteria and anaerobic bacteria can survive in anaerobic conditions, forming stable populations. A key characteristic of facultative anaerobic bacteria is their ability to survive and reproduce in oxygen-free environments, unaffected by ambient oxygen levels. Natural environments contaminated with hydrocarbons, such as soil, groundwater aquifers, freshwater and marine sediments, and oil reservoirs, all share anaerobic characteristics. Facultative anaerobic bacteria can survive and reproduce in these environments. Furthermore, they can utilize hydrocarbon pollutants as carbon and energy sources to remove these pollutants. Therefore, it is worth considering developing a stable population of microorganisms that can remove p-xylene under anaerobic denitrification conditions (Tucci M, et al. Toluene-driven anaerobic biodegradation of chloroformin a continuous-flow bioelectrochemical reactor[J]. Chemosphere, 2023.).

[0007] Existing technology CN101892178A discloses a facultative anaerobic degrading bacterium of benzene series compounds, namely *Microbacterium schleiferi* HBSD-C, which was deposited at the China Center for Type Culture Collection (CCTCC) on November 27, 2009, with accession number CCTCC No. M209284. Colony morphology: pale yellow, round, translucent, with regular, concave edges and a smooth surface; cells are straight rods, Gram-positive, and non-motile; main biochemical characteristics: oxidase-positive, catalase-positive, utilizes glucose, fructose, and sucrose, but not maltose, denitrification-positive, gelatin hydrolysis-negative, facultative anaerobic, and grows at 4℃-41℃. This strain can degrade benzene, toluene, xylene, and mesitylene under facultative anaerobic conditions, and the degradation rate of benzene series compounds at a concentration of approximately 175.8 mg / L is between 25.9% and 41.2% after 3 days. The drawback of this technique is that the denitrification ability of the strains was not measured, and the degradation rate of xylene was low. Summary of the Invention

[0008] This invention addresses the lack of strains capable of efficiently and synergistically removing xylene and nitrogen under anaerobic conditions, providing a *Pseudomonas putida* strain capable of removing both xylene and nitrogen, along with its culture medium, inoculum, and applications. This invention provides a *Pseudomonas putida* strain capable of removing both xylene and nitrogen under anaerobic conditions, with accession number CCTCCNO: M20241686, deposited at the China Center for Type Culture Collection on July 25, 2024. This strain... + -N, NO3 - -N and NO2 - When -N is the sole nitrogen source (15 mg / L), for NH4 + -N, NO3 - -N and NO2 - The removal rates of -N reached 59.08%, 96.90%, and 100%, respectively; when p-xylene was used as the sole carbon source, the 72-hour removal rate of p-xylene by this bacterium reached 55.54%. The optimal denitrification conditions for this bacterium are: optimal carbon source sodium succinate hexahydrate, optimal C / N ratio of 3, optimal culture temperature of 30℃, and optimal pH of 7. It can tolerate weakly alkaline environments but is not tolerant of acidic environments.

[0009] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0010] On the one hand, the present invention provides a *Pseudomonas putida* strain that combines xylene and nitrogen removal, with accession number CCTCC NO: M20241686, which was deposited at the China Center for Type Culture Collection on July 25, 2024, and named *Pseudomonas putida* strain 19, or strain 19 for short, and its 16S rDNA sequence is the sequence shown in SEQ ID NO.1.

[0011] On the other hand, the present invention provides a microbial agent comprising the above-mentioned *Pseudomonas putida* or a preparation thereof.

[0012] Preferably, the preparation of *Pseudomonas putida* includes the above-mentioned *Pseudomonas putida* culture, culture extract, lyophilized powder, fermentation broth, fermentation broth precipitate, fermentation broth supernatant, or fermentation broth extract.

[0013] Preferably, the bacterial agent is prepared by inoculating the above-mentioned *Pseudomonas putida* into a culture medium for fermentation, filtering the fermentation broth to obtain a precipitate, and then freeze-drying it.

[0014] On the other hand, the present invention provides the application of the above-mentioned *Pseudomonas malodorans* or the above-mentioned bacterial agent, the application including denitrification in water or removal of p-xylene in water.

[0015] Preferably, the application includes denitrification in water bodies and synergistic removal of p-xylene.

[0016] On the other hand, the present invention provides a method for denitrification and synergistic removal of paraxylene in water, wherein the method involves adding the above-mentioned *Pseudomonas malodorosa* or the above-mentioned bacterial agent to nitrogen-containing water for cultivation.

[0017] Preferably, the method involves adding the aforementioned *Pseudomonas malodorans* or the aforementioned bacterial agent to nitrogen-containing water for cultivation, which can achieve denitrification and synergistic denitrification of paraxylene in the water under anaerobic conditions.

[0018] Preferably, the nitrogen-containing water body includes wastewater or artificial culture medium, which contains the necessary nutrients for the growth of the aforementioned *Pseudomonas putida* and has suitable pH and temperature conditions.

[0019] Preferably, the nitrogen-containing water body includes a nitrogen-containing culture medium, the components of which include: carbon source, nitrogen source, KH2PO4, Na2HPO4·12H2O, MgSO4·7H2O and trace elements, and the pH of the nitrogen-containing culture medium is 5-9.

[0020] Preferably, the carbon source includes sodium potassium tartrate, sodium succinate hexahydrate, sodium citrate, sodium acetate, or glucose. The carbon source primarily provides energy for the strain's growth and serves as an electron donor for the denitrification process; different carbon sources have a significant impact on the strain's denitrification ability.

[0021] Preferably, the carbon source is sodium succinate hexahydrate.

[0022] Preferably, the nitrogen source includes nitrate, nitrite, or ammonium salt.

[0023] Preferably, the nitrogen source is nitrate.

[0024] Preferably, the carbon-nitrogen molar ratio of the carbon source and the nitrogen source is 1-60.

[0025] Preferably, the carbon-nitrogen molar ratio is 15.

[0026] Preferably, the nitrogen-containing culture medium has a pH of 7.0. Since pH causes changes in cell membrane charge, thus affecting the absorption of nutrients by microorganisms and the activity of related intracellular enzymes, a suitable pH has a significant impact on the metabolic capacity of the strain.

[0027] Preferably, the culture temperature of the nitrogen-containing culture medium is 25-35℃.

[0028] Preferably, the temperature of the nitrogen-containing culture medium is 30°C.

[0029] Preferably, the nitrogen-containing culture medium comprises KNO3 0.100-0.120 g / L, sodium succinate hexahydrate 1.0000-1.2000 g / L, KH2PO4 0.200-0.250 g / L, Na2HPO4·12H2O 0.780-0.800 g / L, MgSO4·7H2O 0.008-0.012 g / L, and trace elements 1.5-2.5 mL / L. The trace elements include FeCl2·4H2O 1.6-2.0 g / L, CoCl2·6H2O 0.23-0.27 g / L, NiCl2·6H2O 0.008-0.012 g / L, CuCl2·2H2O 0.008-0.012 g / L, and MnCl2·4H2O 0.60-

[0030] 0.80g / L, ZnCl2 0.08-0.12g / L, H3BO3 0.4-0.6g / L, Na2MoO4·2H2O 0.02-0.04g / L and NaSeO3·5H2O 0.008-0.012g / L.

[0031] Preferably, the nitrogen-containing culture medium comprises 0.108 g / L KNO3, 1.1562 g / L sodium succinate hexahydrate, 0.226 g / L KH2PO4, 0.792 g / L Na2HPO4·12H2O, 0.01 g / L MgSO4·7H2O, and 2.0 mL / L trace elements, wherein the trace elements include 1.8 g / L FeCl2·4H2O, 0.25 g / L CoCl2·6H2O, 0.01 g / L NiCl2·6H2O, 0.01 g / L CuCl2·2H2O, 0.70 g / L MnCl2·4H2O, 0.1 g / L ZnCl2, 0.5 g / L H3BO3, 0.03 g / L Na2MoO4·2H2O, and NaSeO3·5H2O. 0.01g / L.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] 1. Anaerobic nitrogen-rich wastewater treated with Pseudomonas putida 19 provided by this invention exhibits good anaerobic denitrification capacity under low carbon-to-nitrogen ratio conditions. At the same time, it can effectively remove p-xylene and nitrogen from the water when p-xylene is the only carbon source. Strain 19 shows excellent low carbon-to-nitrogen ratio anaerobic denitrification and good synergistic denitrification capacity for removing p-xylene. The removal rate of p-xylene by this bacterium can reach 55.54% after 72 hours.

[0034] 2. The anaerobic Pseudomonas strain 19 provided by this invention can simultaneously achieve denitrification and paraxylene removal under anaerobic conditions. It is convenient to use, simple to operate, and reduces wastewater treatment costs. This bacterium can be made into a novel microecological agent, showing good application prospects in the treatment of complex wastewater.

[0035] Preservation Instructions

[0036] Bacterial species name: *Pseudomonas putida*;

[0037] Latin name: Pseudomonas putida;

[0038] Strain number: Pseudomonas putida strain 19;

[0039] Preservation institution: China Center for Type Culture Collection;

[0040] Abbreviation for depository institution: CCTCC;

[0041] Address: No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province (within the campus of Wuhan University);

[0042] Deposit date: July 25, 2024;

[0043] Accession number: CCTCC NO: M20241686. Attached Figure Description

[0044] Figure 1 Electron micrographs and Gram staining images of strain 19, where A is an electron micrograph and B is a Gram staining image.

[0045] Figure 2 Phylogenetic tree of strain 19.

[0046] Figure 3 PCR amplification of denitrase-related genes (M is Marker: DL 2000 DNA).

[0047] Figure 4 Growth curve of strain 19 when nitrate is the sole nitrogen source, and NH4+. + -N concentration, NO3 --N concentration, NO2 - -N concentration and TN concentration results graph.

[0048] Figure 5 Growth curve of strain 19 when ammonium salt is the sole nitrogen source, NH4 + -N concentration, NO3 - -N concentration, NO2 - -N concentration and TN concentration results graph.

[0049] Figure 6 Growth curve of strain 19 when nitrite is the sole nitrogen source, NH4 + -N concentration, NO3 - -N concentration, NO2 - -N concentration and TN concentration results graph.

[0050] Figure 7 Effects of different carbon sources on the growth and denitrification of strain 19.

[0051] Figure 8 Effects of different C / N ratios on the growth and denitrification of strain 19.

[0052] Figure 9 Effects of different pH values ​​on the growth and denitrification of strain 19.

[0053] Figure 10 Effects of different temperatures on the growth and denitrification of strain 19.

[0054] Figure 11 Growth curve of strain 19 with p-xylene as the sole carbon source, NH4 + -N concentration, NO3 - -N concentration, NO2 - -N concentration and TN concentration results graph.

[0055] Figure 12 The removal of p-xylene by strain 19 at different culture times. Detailed Implementation

[0056] It is worth noting that the raw materials used in this invention are all commercially available products, and their sources are not specifically limited.

[0057] Culture medium setup:

[0058] DBM (enrichment medium): KNO3 5.0 g / L, sodium succinate hexahydrate 11.1 g / L, KH2PO4 1.0 g / L, Na2HPO4·12H2O 7.03 g / L, MgSO4·7H2O 0.13 g / L, trace elements 2 mL / L, adjust pH to 7.0.

[0059] BTB (bromothymol blue solid medium): KNO3 5.0 g / L, sodium succinate hexahydrate 11.1 g / L, KH2PO4

[0060] 1.0 g / L, Na2HPO4·12H2O 7.03 g / L, MgSO4·7H2O 0.13 g / L, trace elements 2 mL / L, 1% bromothymol blue, 2% agar powder, adjust pH to 7.0.

[0061] Nitrogen-nitrogen medium (C / N = 15:1): KNO3 0.108 g / L, sodium succinate hexahydrate 1.1562 g / L, KH2PO4 0.226 g / L, Na2HPO4·12H2O 0.792 g / L, MgSO4·7H2O 0.01 g / L, trace elements 2 mL / L, pH adjusted to 7.0.

[0062] Nitrite medium (C / N = 15:1): NaNO2 0.0738 g / L, sodium succinate hexahydrate 1.1566 g / L, KH2PO4 0.226 g / L, Na2HPO4·12H2O 0.792 g / L, MgSO4·7H2O 0.01 g / L, trace elements 2 mL / L, pH adjusted to 7.0.

[0063] Ammonia nitrogen medium (C / N = 15:1): NH4Cl 0.058 g / L, sodium succinate hexahydrate 1.1723 g / L, KH2PO4 0.226 g / L, Na2HPO4·12H2O 0.792 g / L, MgSO4·7H2O 0.01 g / L, trace elements 2 mL / L, pH adjusted to 7.0.

[0064] The test medium for carbon removal using p-xylene as the sole carbon source (C / N = 15:1): KNO3 0.108 g / L, KH2PO4

[0065] 0.226 g / L, Na2HPO4·12H2O 0.792 g / L, MgSO4·7H2O 0.01 g / L, trace elements 2 mL / L, adjust pH to 7.0.

[0066] Trace elements: FeCl2·4H2O 1.8g / L, CoCl2·6H2O 0.25g / L, NiCl2·6H2O 0.01g / L, CuCl2·2H2O0.01g / L, MnCl2·4H2O 0.70g / L, ZnCl2 0.1g / L, H3BO3 0.5g / L, Na2MoO4·2H2O0.03g / L, NaSeO3·5H2O 0.01g / L.

[0067] Culture medium sterilization procedure: The culture medium and required materials are sterilized in an autoclave at 121°C for 20 minutes.

[0068] Detection methods and data analysis:

[0069] OD 600 (Indicator biomass), TN, NO3 - -N, NO2 - -N and NH4 + -N content was measured using a spectrophotometer, OD 600 The value was directly measured at a wavelength of 600 nm. TN was determined using the alkaline potassium persulfate oxidation-ultraviolet spectrophotometric method. NO3 was measured... - -N was determined using the aminosulfonic acid-UV spectrophotometric method, NO2 - -N is produced using the sulfonamide-naphthylethylenediamine hydrochloride method, NH4 + -N was determined using Nessler's reagent method.

[0070] The determination of p-xylene followed the method for determining the concentration of residual benzene series compounds in solution as specified in the National Environmental Protection Standard "Determination of Volatile Organic Compounds in Water - Headspace / Gas Chromatography-Mass Spectrometry" (HJ810-2016); the pure p-xylene standard was diluted to a concentration of 100 mg·L⁻¹ using chromatographic grade methanol. -1 and 1000 mg·L -1 The stock solution was prepared by diluting pure toluene-D8 standard to 1000 mg·L⁻¹. -1 Prepare a stock solution for later use. Using a microsyringe, transfer the p-xylene stock solution into a headspace vial containing 10 mL of matrix-modified solution to prepare a series of p-xylene concentrations of 50, 100, 160, 200, 250, and 300 μg·L⁻¹. -1 The solution, with the internal standard toluene-D8 concentration fixed at 200 μg·L⁻¹, was prepared. -1 Then, instrumental analysis was performed using GC-MS, and the results showed that p-xylene was available in the range of 0–300 μg·L⁻¹. -1 Good linearity across the concentration range (R) 2 >0.99) and the method detection limit is less than 1 μg·L -1A GC-MS system was used, with the following headspace sampling system parameters: furnace temperature 80℃, sampling needle temperature 95℃, transfer line temperature 110℃, carrier gas pressure (He) 19.0 psi, equilibration time 30 min, pressurization time 1 min, injection time 0.04 min, needle withdrawal time 0.5 min, and GC cycle time 33 min. A DB-1 column (60 m × 0.32 mm × 1.0 μm) was used, with high-purity helium as the carrier gas. The GC temperature program was as follows: initial temperature 60℃, hold for 2 min; then increase to 120℃ at 5℃ / min, hold for 3 min; then increase to 230℃ at 20℃ / min, hold for 3 min, for a total program of 25 min. The MS conditions were: electron impact (EI) ion source; ion source temperature 230℃, ionization energy 70 eV, and interface temperature 280℃. Scanning mode: Full scan; Scan range: 35 amu to 300 amu.

[0071] Example 1: Identification of the strain

[0072] (1) Enrichment: Take 5g of sediment and add it to 100mL of sterile DBM medium. Place the mixture in an anaerobic bag and incubate at 30℃. Transfer 5mL of the culture medium to fresh sterile DBM medium and repeat the enrichment process for 3 rounds.

[0073] (2) Separation: Dilute the enriched solution sequentially according to a gradient (10) -1 ~10 -7 Take 10 mL of the solution and spread it onto BTB solid medium. Place the medium in an anaerobic bag and incubate at 30°C until distinct single colonies appear. Pick the blue-turned single colonies, purify them by streak multiple times, and store them at 4°C.

[0074] (3) Screening: The isolated and purified bacterial strains were inoculated into nitrate nitrogen medium, placed in anaerobic bags, and incubated statically in a 30°C incubator. After 24 hours, the change in nitrate nitrogen concentration was measured, and strain No. 19 with the highest nitrate nitrogen removal rate was obtained through screening.

[0075] (4) Gram staining: Bacterium 19 was inoculated into nitrate medium and incubated statically at 30°C for 24 hours, with OD monitored. 600 After the bacteria reached the logarithmic growth phase, Gram staining was performed to confirm that it was a Gram-negative strain (staining results are shown in [reference needed]). Figure 1 (B).

[0076] (5) Biological identification:

[0077] After activating strain 19 by streak plating, it was diluted and spread onto BTB plates. Colony morphology was observed; the colonies were round, transparent, moist, and smooth. Scanning electron microscopy was used to observe the cell morphology of strain 19, and the results are as follows: Figure 1As shown in Figure A, the bacterial cells are rod-shaped, with a size of (0.9–0.92 μm) × 3.92 μm. Bacterial DNA was extracted using a DNA extraction kit, and PCR was performed using universal 16S rRNA primers to obtain the 16S rRNA fragment, which was then sequenced. The 16S rDNA sequence of strain 19 was 1431 bp in length, as shown in SEQ ID NO. 1. Blast analysis of the sequencing results on NCBI showed 99% homology with *Pseudomonas putida* strain BBAL5-01 (Gene Bank No.: FJ217182.1). Phylogenetic analysis showed that strain 19 clustered with *Pseudomonas putida* BBAL5-01, as shown in Figure A. Figure 2 Based on morphological characteristics, physiological and biochemical properties, and molecular biological identification features, strain 19 was identified as belonging to *Pseudomnas putida*. This bacterium was deposited at the China Center for Type Culture Collection on July 25, 2024, with the accession number CCTCC NO: M20241686, and was named *Pseudomnas putida* strain 19, or simply strain 19.

[0078] (6) PCR amplification of denitrase-related genes:

[0079] The narG, nirS, norR, and nosZ genes of strain 19 were verified by PCR. Specific primers and amplification conditions are shown in Tables 1 and 2, respectively. PCR products were analyzed by 1% agarose gel electrophoresis and EB staining. Results are shown in [Table 1]. Figure 3 This indicates that strain 19 contains denitrase-related genes narG, nirS, norR, and nosZ in its genome.

[0080] The amplification system was as follows: the total reaction volume was 25 μL (10 μL ddH2O, 12.5 μL 2×PCR Mix, 1 μL upstream primer, 1 μL downstream primer, and 1 μL bacterial template DNA).

[0081] Table 1 Primers for amplification of denitrase-related genes

[0082]

[0083]

[0084] Table 2 Amplification procedures for denitrase-related genes

[0085]

[0086] Example 2: Denitrification characteristics of the strain

[0087] Strain activation: Inoculate the frozen strain into a 50mL centrifuge tube containing 50mL of nitrate medium, seal the tube with sealing film, and incubate statically at 30℃. Monitor the OD of the bacterial culture. 600 Once the bacterial culture has grown to the logarithmic phase, pour 10 mL of the culture into a 50 mL centrifuge tube, add 40 mL of nitrate culture medium, seal the tube tightly with sealing film, and culture it again until the logarithmic phase before use.

[0088] The activated strain 19 was inoculated into 50 mL centrifuge tubes at an inoculation rate of 2% v / v into 50 mL of nitrate nitrogen medium (containing NO3). - -N as nitrogen source), nitrite nitrogen culture medium (50 mL, with NO2) - (NH4+ as nitrogen source) and ammonia nitrogen culture medium (50 mL, with NH4+ as nitrogen source) + In a nitrogen source (N-N), the bottle mouth is sealed with film and incubated statically in a 30℃ incubator for 48 hours. OD is measured every 6 hours. 600 Value, NH4 + -N concentration, NO3 - -N concentration, NO2 - -N concentration and TN concentration.

[0089] By selecting different inorganic nitrogen sources NO3 - -N, NH4 + -N and NO2 - -N were used as the sole nitrogen source to study the nitrogen removal capacity of strain 19. When NO3- - -N is used as the sole nitrogen source (see...) Figure 4 The growth curve of the strain initially increased within 0–48 h, then gradually stabilized, reaching a maximum value of 0.031 at 36 h; simultaneously, within 18 h, NO3… - The NO-N concentration decreased from the initial 15 mg / L to 0.48 mg / L, with a removal rate of 0.81 mg / (L·h) and a removal rate of 96.90%. No NO2 was detected during this process. - -N and NH4 + -N accumulation, NO2 - -N reached a maximum concentration of 6.51 mg / L, but was completely degraded at the next time point, with a TN degradation rate of 92.48%. When NH4... + -N (see Figure 5 ) and NO2 - -N (see Figure 6 When NH4 is used as the sole nitrogen source, + -N and NO2 - The removal rates of -N were 59.08% and 100%, respectively, with removal rates reaching 0.48 mg / (L·h) and 0.83 mg / (L·h), and the removal rate was mainly NH4+. +When nitrogen (N) was the sole nitrogen source, almost no nitrite accumulation occurred during the incubation process, and the TN degradation rates were 20.17% and 88.97%, respectively. These results indicate that strain 19 exhibits highly efficient inorganic nitrogen removal capabilities and has the potential for enhanced application in high-nitrogen-rich wastewater treatment. Furthermore, the amplification of the narG, nirS, norR, and nosZ genes in Example 1 further demonstrates the anaerobic denitrification potential of this strain.

[0090] Example 3: Effects of carbon source, C / N ratio, pH, and temperature on anaerobic denitrification of the strain

[0091] The strain activation was carried out in accordance with the method described in Example 2.

[0092] The activated bacterial culture was inoculated at a rate of 2% v / v into 50 mL centrifuge tubes containing 50 mL of nitrate-nitrogen medium. Based on the nitrate-nitrogen medium, single-factor optimizations were performed according to the following settings: For carbon source optimization, different carbon sources were selected: potassium sodium tartrate, sodium succinate hexahydrate, sodium citrate, sodium acetate, and glucose, all at a concentration of 225 mg / L; for C / N ratio optimization, the KNO3 concentration was kept constant, and different C / N ratios were set at 1, 3, 5, 10, 15, 30, and 60; for pH optimization, different pH values ​​were set at 5, 7, and 9; for culture temperature optimization, different temperatures were set at 25℃, 30℃, and 35℃. Each group was set up in triplicate. After static incubation for 48 h, the culture tubes were removed and OD was measured. 600 and NO3 - The nitrogen removal capacity of the strain was investigated by considering four factors: carbon source, C / N ratio, temperature, and pH.

[0093] The effects of different carbon sources on the growth and denitrification of strain 19 are shown in the figure. Figure 7 The strain could grow under all five carbon sources. When glucose was used as the sole carbon source, strain 19 achieved its highest growth rate of 0.1278, but at this time, its nitrogen removal rate was only 96.01%. However, when sodium succinate hexahydrate was used as the carbon source, the nitrate removal rate reached its maximum of 97.85%. The effect of different C / N ratios on the nitrogen removal capacity of strain 19 is as follows: Figure 8 As shown, with the increase of C / N, the OD of the strain... 600 It is also constantly increasing; when C / N reaches 15, OD 600The nitrogen removal efficiency of the strain reached a maximum of 0.036 and then decreased. With increasing C / N ratio, the nitrogen removal efficiency first increased and then decreased, reaching a maximum of 99.11% at a C / N ratio of 3. Furthermore, the nitrate removal rate of this strain was above 80% across a C / N ratio range of 1–60. Especially at a C / N ratio of 1, the strain still maintained a high nitrate removal rate (96.53%). Given that strain 19 exhibits the highest nitrogen removal efficiency at a C / N ratio of 3, this effectively addresses the problem of insufficient carbon source and low nitrogen removal efficiency in anaerobic denitrifying bacteria treating wastewater with lower C / N ratios. The nitrogen removal capacity of strain 19 at different pH values ​​is shown below. Figure 9 As shown, the strain barely grows at pH 5, but can grow at pH 7–9, with optimal growth at pH 7. Furthermore, its nitrogen removal capacity consistently reaches 90%, indicating that this strain can only tolerate neutral and slightly alkaline environments and cannot tolerate acidic environments. Different culture temperatures result in varying nitrate removal capacities, as shown in the results. Figure 10 As shown, strain 19 can grow under culture conditions ranging from 25 to 35°C, with the best growth observed at 35°C. (OD value missing) 600 The value was 0.048, but the lowest nitrate removal rate was 82.67%. Strain 19 maintained a high removal rate at temperatures ranging from 25℃ to 35℃, reaching over 80% and up to 96%, indicating that this strain can tolerate a wide temperature range.

[0094] Example 4: Removal characteristics of the strain of para-xylene

[0095] The strain activation was carried out in accordance with the method described in Example 2.

[0096] The activated bacterial strain was inoculated at a rate of 5% v / v into a 100 mL anaerobic flask containing 50 mL of p-xylene as the sole carbon source for the test medium. Argon gas was passed through the flask for 4 minutes to create an anaerobic environment. After aeration, the flask opening was sealed with sealing film. 10 μL of p-xylene solution was injected using a microsyringe to achieve a final concentration of 160 mg / L. The flask was then incubated at 30°C for 160 rpm for 72 h. Three parallel experiments were conducted; OD was measured every 12 h. 600 Value, NH4 + -N concentration, NO3 - -N concentration, NO2 - -N concentration, TN concentration, and p-xylene concentration.

[0097] The changes in the concentration of each component during the removal of p-xylene by strain 19 are as follows: Figure 11 and Figure 12 As shown, when p-xylene was used as the sole carbon source, the growth curve of the strain gradually decreased from 0 to 72 h, reaching a minimum of 0.025 at 72 h. Simultaneously, within 72 h, NO3...- The concentration of NO2- gradually decreased over time, from an initial 17 mg / L to 2.40 mg / L, with a removal rate of 0.20 mg / (L·h) and a removal rate of 89.70%. - The concentration of -N increases with time, reaching a peak of 12.29 mg / L at 72 hours. No NH4+ was present during this process. + -N accumulation and TN removal rate were 10.93% (see...) Figure 11 The p-xylene content also gradually decreased over time, from an initial 160 mg / L to 70.96 mg / L, with a removal rate of 1.24 mg / (L·h) and a removal rate of 55.54% (see...). Figure 12 ).

[0098] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A *Pseudomonas putida* strain capable of removing both xylene and nitrogen (… Pseudomonas putida ), characterized in that, The preservation number of the bacterium is CCTCC NO: M20241686, which has been preserved in China Center for Type Culture Collection on July 25, 2024.

2. An inoculant characterized in that, The bacterium comprises the Pseudomonas putida according to claim 1 or a preparation of the Pseudomonas putida according to claim 1; the preparation of the Pseudomonas putida comprises a culture, a freeze-dried powder, a fermentation broth or a fermentation broth precipitate of the Pseudomonas putida.

3. Use of the P. putida according to claim 1 or of the bacterial agent according to claim 2, characterized in that The application is anaerobic denitrification in water or removal of p-xylene in water.

4. A method for denitrification and removal of p-xylene in a water body, characterized in that, The method comprises adding the Pseudomonas putida according to claim 1 or the bacterium according to claim 2 into a nitrogen-containing water body for culture, so as to realize denitrification and removal of p-xylene in water in an anaerobic environment.

5. The method of claim 4, wherein, The nitrogen-containing water body comprises a nitrogen-containing culture medium, and components of the nitrogen-containing culture medium comprise a carbon source, a nitrogen source, KH2PO4, Na2HPO4·12H2O, MgSO4·7H2O and trace elements, and the pH of the nitrogen-containing culture medium is 5-9.

6. The method of claim 5, wherein, The carbon source comprises potassium sodium tartrate, sodium succinate hexahydrate, sodium citrate, sodium acetate or glucose.

7. The method of claim 6, wherein, The carbon source comprises sodium succinate hexahydrate.

8. The method of claim 5, wherein, The nitrogen source comprises nitrate, nitrite or ammonium salt.

9. The method of claim 8, wherein, The nitrogen source is nitrate.

10. The method of claim 5, wherein, The carbon-nitrogen molar ratio of the carbon source and the nitrogen source is 1-60.

11. The method of claim 10, wherein, The carbon-nitrogen molar ratio is 15.

12. The method of claim 5, wherein, The pH is 7.

0.

13. The method of claim 5, wherein, The culture temperature of the nitrogen-containing culture medium is 25-35°C.

14. The method of claim 13, wherein, The culture temperature is 30°C.

15. The method of claim 5, wherein, The components of the nitrogen-containing culture medium comprise KNO3 0.100-0.120 g / L, sodium succinate hexahydrate 1.0000-1.2000 g / L, KH2PO4 0.200-0.250 g / L, Na2HPO4·12H2O 0.780-0.800 g / L, MgSO4·7H2O 0.008-0.012 g / L and trace elements 1.5-2.5 mL / L, and the trace elements comprise FeCl2·4H2O 1.6-2.0 g / L, CoCl2·6H2O 0.23-0.27 g / L, NiCl2·6H2O 0.008-0.012 g / L, CuCl2·2H2O 0.008-0.012 g / L, MnCl2·4H2O 0.60-0.80 g / L, ZnCl2 0.08-0.12 g / L, H3BO3 0.4-0.6 g / L, Na2MoO4·2H2O 0.02-0.04 g / L and NaSeO3·5H2O 0.008-0.012 g / L.

16. The method of claim 15, wherein, The components of the nitrogen-containing medium include KNO3 0.108 g / L, sodium succinate hexahydrate 1.1562 g / L, KH2PO4 0.226 g / L, Na2HPO4-12H2O 0.792 g / L, MgSO4-7H2O 0.01 g / L, and trace elements 2.0 mL / L, which include FeCl2-4H2O 1.8 g / L, CoCl2-6H2O 0.25 g / L, NiCl2-6H2O 0.01 g / L, CuCl2-2H2O 0.01 g / L, MnCl2-4H2O 0.70 g / L, ZnCl2 0.1 g / L, H3BO3 0.5 g / L, Na2MoO4-2H2O 0.03 g / L, and NaSeO3-5H2O 0.01 g / L.

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